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Green synthesis of silver nanoparticles usingAzadirachta indica aqueous leaf extract

Shakeel Ahmed, Saifullah, Mudasir Ahmad, Babu Lal Swami,Saiqa Ikram*

Department of Chemistry, Faculty of Natural Sciences, Jamia Millia Islamia (central university), New Delhi 110025,

India

a r t i c l e i n f o

Article history:

Received 25 March 2015

Received in revised form

29 May 2015

Accepted 15 June 2015

Available online 26 June 2015

Keywords:

Green synthesis

Silver nanoparticle

Azadirachta indica

Bioreduction

Plant extract

* Corresponding author. Tel.: þ91 11 2698171E-mail addresses: shakeelchem11@gmail

Peer review under responsibility of The Ehttp://dx.doi.org/10.1016/j.jrras.2015.06.0061687-8507/Copyright© 2015, The Egyptian Socopen access article under the CC BY-NC-ND l

a b s t r a c t

In this study, rapid, simple approach was applied for synthesis of silver nanoparticles using

Azadirachta indica aqueous leaf extract. The plant extract acts both as reducing agent as

well as capping agent. To identify the compounds responsible for reduction of silver ions,

the functional groups present in plant extract were investigated by FTIR. Various tech-

niques used to characterize synthesized nanoparticles are DLS, photoluminescence, TEM

and UVeVisible spectrophotometer. UVeVisible spectrophotometer showed absorbance

peak in range of 436e446 nm. The silver nanoparticles showed antibacterial activities

against both gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) mi-

croorganisms. Photoluminescence studies of synthesised silver nanoparticles were also

evaluated. Results confirmed this protocol as simple, rapid, one step, eco-friendly, non-

toxic and an alternative conventional physical/chemical methods. Only 15 min were

required for the conversion of silver ions into silver nanoparticles at room temperature,

without the involvement of any hazardous chemical.

Copyright © 2015, The Egyptian Society of Radiation Sciences and Applications. Production

and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

The ‘green’ environment friendly processes in chemistry and

chemical technologies are becoming increasingly popular and

are much needed as a result of worldwide problems associ-

ated with environmental concerns (Thuesombat,

Hannongbua, Akasit, & Chadchawan, 2014). Silver is the one

of the most commercialised nano-material with five hundred

tons of silver nanoparticles production per year (Larue et al.,

2014) and is estimated to increase in next few years.

7x3255..com (S. Ahmed), sikram@

gyptian Society of Radiat

iety of Radiation Sciencesicense (http://creativecom

Including its profound role in field of high sensitivity bio-

molecular detection, catalysis, biosensors and medicine; it is

been acknowledged to have strong inhibitory and bactericidal

effects along with the anti-fungal, anti-inflammatory and

anti-angiogenesis activities (El-Chaghaby & Ahmad, 2011;

Veerasamy et al., 2011).

A number of techniques are available for the syntheses of

silver nanoparticles like ion sputtering, chemical reduction,

sol gel, etc. (Bindhu&Umadevi, 2015; Mahdi, Taghdiri, Makari,

& Rahimi-Nasrabadi, 2015; Padalia, Moteriya, & Chanda, 2014;

Sre, Reka, Poovazhagi, Kumar, & Murugesan, 2015);

jmi.ac.in (S. Ikram).

ion Sciences and Applications.

andApplications. Production and hosting by Elsevier B.V. This is anmons.org/licenses/by-nc-nd/4.0/).

J o u r n a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1e72

unfortunately many of the nanoparticle syntheses methods

involve the use of hazardous chemicals or high energy re-

quirements, which are rather difficult and including wasteful

purifications (Ahmed, Ahmad, Swami, & Ikram, 2015). Thus; a

scenario is that whatever the method followed, will always

leading to the chemical contaminations during their synthe-

ses procedures or in later applications with associated limi-

tations. Yet; one cannot deny their ever growing applications

in daily life. For instances; “The Noble Silver Nanoparticles”

are striving towards the edge-level utilities in every aspect of

science and technology including the medical fields; thus

cannot be neglected just because of their source of generation.

Hence, it is becoming a responsibility to emphasise on an

alternate as the synthetic route which is not only cost effec-

tive but should be environment friendly in parallel. Keeping in

view of the aesthetic sense, the green syntheses are rendering

themselves as key procedure and proving their potential at

the top. The techniques for obtaining nanoparticles using

naturally occurring reagents such as sugars, biodegradable

polymers (chitosan, etc.), plant extracts, and microorganisms

as reductants and capping agents could be considered

attractive for nanotechnology (Ahmed, Ahmad,& Ikram, 2014;

Ahmed & Ikram, 2015; Kharissova, Dias, Kharisov, P�erez, &

P�erez, 2013). Greener syntheses of nanoparticles also pro-

vides advancement over other methods as they are simple,

one step, cost-effective, environment friendly and relatively

reproducible and often results in more stable materials

(Mittal, Batra, Singh, & Sharma, 2014). Microorganisms can

also be utilized to produce nanoparticles but the rate of syn-

theses are slow compared to routes involving plants mediated

synthesis (Ahmed et al., 2015). Although, the potential of

higher plants as source for this purpose is still largely unex-

plored. Very recently plant extract of marigold flower (Padalia

et al., 2014), Ziziphora tenuior (Sadeghi & Gholamhoseinpoor,

2015), Abutilon indicum (Ashokkumar, Ravi, & Velmurugan,

2013), Solanum tricobatum (P. Logeswari, Silambarasan, &

Abraham, 2013), Erythrina indica (Sre et al., 2015), beet root

(Bindhu & Umadevi, 2015), mangosteen (Veerasamy et al.,

2011), Ocimum tenuiflorum (Peter Logeswari, Silambarasan, &

Abraham, 2012), Spirogyra varians (Salari, Danafar, Dabaghi,

& Ataei, 2014), Melia dubia (Ashokkumar et al., 2013), olive

(Khalil, 2013), leaf extract of Acalypha indica with high anti-

bacterial activities (Krishnaraj et al., 2010) and of Sesuvium

portulacastrum also reported in literature with nanoparticle

size ranging from 5 to 20 nm (Nabikhan, Kandasamy, Raj, &

Alikunhi, 2010) are brimming in literature as a source for the

synthesis of silver nanosilver particles as an alternative to the

conventional methods.

Considering the vast potentiality of plants as sources this

work aims to apply a biological green technique for the syn-

thesis of silver nanoparticles as an alternative to conventional

methods. In this regard, leaf extract of Azadirachta indica

(commonly known as neem) a species of familyMeliaceaewas

used for bioconversion of silver ions to nanoparticles. This plant

is commonly available in India and each part of this tree has

been used as a household remedy against various human

ailments from antiquity and for treatment against viral, bac-

terial and fungal infections (Omoja et al., 2011). Silver nano-

particles can be produced at low concentration of leaf extract

without using any additional harmful chemical/physical

methods. The effect of concentration of metal ions and con-

centration of leaf extract quantity were also evaluated to

optimize route to synthesise silver nanoparticle. The method

applied here is simple, cost effective, easy to perform and

sustainable.

2. Experimental

Typically, a plant extract-mediated bioreduction involves

mixing the aqueous extract with an aqueous solution of the

appropriatemetal salt. The synthesis of nanoparticle occurs at

room temperature and completes within a few minutes.

2.1. Preparation of plant extract

A. indica leaf extract was used to prepare silver nanoparticles

on the basis of cost effectiveness, ease of availability and its

medicinal property. Fresh leaves were collected from univer-

sity campus in month of February. They were surface cleaned

with running tap water to remove debris and other contami-

nated organic contents, followed by double distilled water and

air dried at room temperature. About 20 gm of finely cut leaves

were kept in a beaker containing 200mL double distilledwater

and boiled for 30 min. The extract was cooled down and

filteredwithWhatman filter paper no.1 and extract was stored

at 4 �C for further use.

2.2. Green synthesis of silver nanoparticles

Silver nitrate GR used as such (purchased from Merck, India).

100 mL, 1 mM solution of silver nitrate was prepared in an

Erlenmeyer flask. Then 1, 2, 3, 4 and 5 mL of plant extract was

added separately to 10 mL of silver nitrate solution keeping its

concentration at 1 mM. Silver nanoparticles were also syn-

thesized by varying concentration of AgNO3 (1 mMe5 mM)

keeping extract concentration constant (1 mL). This setup was

incubated in a dark chamber to minimize photo-activation of

silver nitrate at room temperature. Reduction of Agþ to Ag0

was confirmed by the colour change of solution from colour-

less to brown. Its formation was also confirmed by using

UVeVisible spectroscopy.

2.3. Characterization of synthesised silver nanoparticles

UVeVis spectral analysis was done by using Shimadzu

UVevisible spectrophotometer (UV-1800, Japan). UVeVisible

absorption spectrophotometer with a resolution of 1 nm

between 200 and 800 nm was used. One millilitre of the

sample was pipetted into a test tube and subsequently

analysed at room temperature. Dynamic light scattering

(Spectroscatter 201) was used to determine the average size

of synthesized silver nanoparticles. FTeIR spectra of were

recorded on Perkin Elmer 1750 FTIR Spectrophotometer. The

particle size and surface morphology was analysed using

Transmission electron microscopy (TEM), operated at an

accelerated voltage of 120 kV. Photoluminescence studies

were evaluated by using eclipse Fluorescence spectropho-

tometer (agilent technologies).

J o u r n a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1e7 3

2.4. Fixation of different parameters

The reaction was monitored at different time intervals. The

reactionwasmonitored using different concentration of silver

nitrate (1 mM, 2 mM, 3 mM, 4 mM and 5 mM) and also by

varying leaf extract solution (1e5 mL) and their absorbance

was measured.

2.5. Assessment of antimicrobial assay

The antibacterial assays were done on human pathogenic

Escherichia coli and Staphylococcus aureus by using standard disc

diffusion method. Mackonkey broth (HiMedia) medium was

used to sub culture bacteria and were incubated at 37 �C for

24 h. Fresh overnight cultures were taken and spread on the

Mackonkey agar plates to cultivate bacteria. Sterile paper

discs of 5 mm diameter saturated with plant extract, silver

nanoparticle and double distilled water (as control) were

placed in each plate and incubated again at 37 �C for 24 h and

the antibacterial activity was measured based on the inhibi-

tion zone around the disc impregnated with plant extract and

synthesized silver nanoparticle.

3. Results and discussion

3.1. Visual observation and UVeVis spectroscopy

In all experiments, addition of plant extract of A. indica into

the beakers containing aqueous solution of silver nitrate led to

the change in the colour of the solution to yellowish to reddish

brown (shown in Fig. 1) within reaction duration due to exci-

tation of surface plasmon vibrations in silver nanoparticles

(Veerasamy et al., 2011). On addition of different concentra-

tion (1e5mL) of leaf extracts to aqueous silver nitrate solution

keeping its concentration 10mL (1mM) constant, the colour of

the solution changed from faint light to yellowish brown and

finally to colloidal brown indicating formation of silver

nanoparticles. Different parameters were optimized including

concentration of silver nitrate and A. indica leaf extract, and

time which had been identified as factors affecting the yields

of silver nanoparticles. Silver nanoparticles were synthesized

at different concentrations of leaf extract such as 1e5 mL

using 1 mM of silver nitrate were analysed by UV spectra of

Plasmon resonance band observed at 436e446 nm similar to

those reported in literature (Obaid, Al-Thabaiti, Al-Harbi, &

Fig. 1 e Digital optical images of synthesized silver

nanoparticles with different conc (1e5 mM) of AgNO3.

Khan, 2015). If we increase the leaf extract concentration to

4 mL, there is increase in wavelength up to 448 nm as pre-

sented in Fig. 2a. The slight variations in the values of absor-

bance signifies that the changes are the particle size (Tripathy,

Raichur, Chandrasekaran, Prathna, & Mukherjee, 2010). On

increasing concentration of extract there is increase in in-

tensity of absorption. The UVeVisible spectra recorded after

different time intervals of 1 h, 2 h, 3 h, 4 h, 18 h and 24 h from

the initiation of reaction with varying amount of plant extract

Fig 3aee. It is generally recognize that UVeVis spectroscopy

could be used to examine size and shape-controlled nano-

particles in aqueous suspensions.

Parallel changes in colour have been observed when

different concentrations (1 mMe5 mM) of silver nitrate was

used by keeping plant extract (1mL) constant. The appearance

of the brown colour was due to the excitation of the Surface

Plasmon Resonance (SPR), typical of silver nanoparticles

having absorbance values which were reported earlier in the

visible range of 446e448 nm (Banerjee, Satapathy,

Mukhopahayay, & Das, 2014; Tripathy et al., 2010). There is

increase in intensity of absorption peaks after regular in-

tervals of time and the colour intensity increased with the

duration of incubation. It was also observed from Fig. 2b that

the intensity of absorption peaks increases with increase in

the concentration of the silver nitrate salt. All the results are

very close already reported in literature showing absorbance

at 445 nm of silver nanoparticles synthesized by Cochlo-

spermum religiosum extract (Sasikala, Linga Rao, Savithramma,

& Prasad, 2014) and by Pithophoraoe dogonia extract (Sinha,

Paul, Halder, Sengupta, & Patra, 2014). The UVevis spectra

recorded, implied that most rapid bioreduction was achieved

using A. indica leaf extract as reducing agent. The UVevis

spectra and visual observation revealed that formation of

silver nanoparticles occurred rapidly within 15 min.

3.2. Particle size and distribution

The size distribution histogram of dynamic light scattering

(DLS) indicates that the size of these silver nanoparticles is

34 nm. Some distribution at lower range of particle size in-

dicates that the synthesized particles are also in lower range

of particle size. Fig. 4 shows the DLS pattern of the suspension

of Ag nanoparticles synthesized using A. Indica aqueous leaf

extract.

3.3. FTIR analysis

The dual role of the plant extract as a reducing and capping

agent and presence of some functional groups was confirmed

by FTIR analysis of silver nanoparticle. A broad band between

3454 cm�1 is due to the NeH stretching vibration of group NH2

and OH the overlapping of the stretching vibration of attrib-

uted for water andA. indica leaf extractmolecules. The band at

1636 cm�1 corresponds to amide C]O stretching and a peak at

2083 cm�1 can be assigned to alkyne group present in phyto-

constituents of extract Fig. 5. The observed peaks at

1113 cm�1 denote eCeOC- linkages, or eCeO- bonds. The

observed peaks are mainly attributed to flavanoids and ter-

penoids excessively present in plants extract (Banerjee et al.,

2014; Prathna, Chandrasekaran, Raichur, & Mukherjee, 2011).

Fig. 2 e UVeVis spectra showing absorbance with different conc. of (a) plant extract (1e5 mL) and (b) AgNO3 (1e5 mM).

Fig. 3 e UVeVis spectra showing absorbance with (different time intervals) with conc. of (a) 1 mL extract (b) 2 mL extract (c)

3 mL extract (d) 4 mL extract (e) 5 mL extract) and (f) extract, AgNO3 solution and silver nanoparticle.

J o u r n a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1e74

Fig. 4 e DLS histogram and TEM image of synthesised silver nanoparticles.

Fig. 5 e FTIR spectra of plant extract and synthesised silver nanoparticle.

Table 1 e Zone of inhibition (mm) obtained by discdiffusion method.

Components Zone of inhibition (mm)

E. coli S. aureus

Control NZ NZ

Plant extract NZ NZ

Silver nanoparticle 9 9

J o u r n a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1e7 5

On the other hand, the extract sample prepared shows a wide

and strong peak with maximum intensity at 553 cm�1. The

results are in good agreement with those found in literature

(Mahdi et al., 2015). From FTeIR results, it can be concluded

that some of the bioorganics compounds fromA. indica extract

formed a strong coating/capping on the nanoparticles.

3.4. TEM analysis

Transmission electron microscopy (TEM) has been used to

identify the size, shape and morphology of nanoparticles. It

reveals that the silver nanoparticles are well dispersed and

predominantly spherical in shape, while some of the NPswere

found to be having structures of irregular shape as shown in

Fig. 4. The nanoparticles are homogeneous and spherical

which conforms to the shape of SPR band in the UVevisible

spectrum. The particle size agrees with that calculated from

DLS histogram with average diameter of around 34 nm.

3.5. Antimicrobial activity

Silver nanoparticles, due to their antimicrobial properties

have been used most widely in the health industry, medicine,

textile coatings, food storage, dye reduction, wound dressing,

antiseptic creams and a number of environmental applica-

tions (Gao et al., 2014). Since ancient times, elemental silver

and its compounds have been used as antimicrobial agents;

and was used to preserve water in form of silver coins/silver

vessels (Devi & Joshi, 2015; Padalia et al., 2014). We have

examined A. indica extract mediated silver nanoparticles as

possible antibacterial agents. The plant extract and those

mediated silver nanoparticles were immediately tested for

respective antimicrobial activities towards both gram positive

(S. aureus) and gram negative (E. coli) bacterial strains showing

the zones of inhibition. Based on the zone of inhibition pro-

duced, synthesized silver nanoparticles prove to exhibit good

antibacterial activity against E. coli and S. aureus. On the other

hand, control and plant extract alone did not exhibit any

antibacterial activity. Although, it is to be presumed that the

leaves extract of the plant used possess the antibacterial ac-

tivities and must be reflected through greater inhibition zone

but it alone shows very low activity due to its medium of

extraction as well as lower concentration during experimen-

tation. The results of antibacterial activities of prepared silver

nanoparticles evaluated from the disc diffusion method are

given in Table 1. The silver nanoparticles showed efficient

Fig. 6 e Fluorescence spectra of silver nanoparticles formed with excitation at (a) 280 nm and (b) 300 nm.

J o u r n a l o f R a d i a t i o n R e s e a r c h and A p p l i e d S c i e n c e s 9 ( 2 0 1 6 ) 1e76

antimicrobial property compared to other due to their

extremely large surface area providing better contact with cell

wall of microorganisms (Ibrahim, 2015).

3.6. Photoluminescence study

Silver nanoparticles are reported to exhibit visible photo-

luminescence and their fluorescence spectra are shown in

Fig. 6. The silver nanoparticles were found to be luminescent

with two emissions at 280 and 561 nm for an excitation at

280 nm. When nanoparticles were excited at 300 nm, it

showed two excitation at 300 and 600 nm, the excitation at

300 nm is of high intensity in comparison to other one. The

luminescence at 280 and 300 nm may be due to presence of

biochemical or antioxidants present in plant extract. The

nanoparticles synthesised using olive leaf extract are also

reported to be luminescent with emission band at 425 nm

(Khalil, Ismail, & El-Magdoub, 2012).

4. Conclusion

A simple one-pot green synthesis of stable silver nano-

particles using A. indica leaf extract at room temperature was

reported in this study. Synthesis was found to be efficient in

terms of reaction time as well as stability of the synthesized

nanoparticles which exclude external stabilizers/reducing

agents. It proves to be an eco-friendly, rapid green approach

for the synthesis providing a cost effective and an efficient

way for the synthesis of silver nanoparticles. Therefore, this

reaction pathway satisfies all the conditions of a 100% green

chemical process. The synthesised silver nanoparticles

showed efficient antimicrobial activities against both E. coli

and S. aureus. Benefits of using plant extract for synthesis is

that it is energy efficient, cost effective, protecting human

health and environment leading to lesser waste and safer

products. This eco-friendly method could be a competitive

alternative to the conventional physical/chemical methods

used for synthesis of silver nanoparticle and thus has a po-

tential to use in biomedical applications and will play an

important role in opto-electronics andmedical devices in near

future.

Acknowledgement

Author SA sincerely acknowledges the financial support from

University Grant Commission (UGC), New Delhi in form of

Junior Research Fellowship (JRF). SI sincerely thanks to Jamia

Millia Islamia for its financial support (grant no.AC-6(15)/RO-

2014).

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